Pharmaceutical composition for prevention or treatment of cognitive function disorders comprising spinosyn

ABSTRACT

The pharmaceutical composition of the present invention, which comprises spinosin or a spinosin-containing herbal extract, is a composition for preventing and treating cognitive function disorders, and can effectively improve memory and learning ability, and effectively treat and prevent cognitive function disorders such as dementia and amnesia.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition comprisingspinosin for prevention and treatment of cognitive impairments. Moreparticularly, the present invention relates to a pharmaceuticalcomposition which can improve memory and learning ability, thuseffectively preventing and treating disorders such as dementia andamnesia.

BACKGROUND ART

With the development of medical technology and the improvement of livingstandards, the average lifespan of a human has been almost doubled overthe past half century, and thus the ratio of the elderly population tothe total population has increased rapidly. Along with the developmentof an aging society, senile dementia has emerged as one of the majorhealth problems that need to be addressed in the twenty-first century.Therefore, the need to develop functional materials, foods, etc. thatcan prevent and treat cognitive impairments including dementia has beenincreasing.

Dementia that is a typical disease of cognitive impairments is apathological condition that needs to be distinguished from normal agingand is classified into Alzheimer's disease, vascular dementia, and otherdementias caused by alcoholism, trauma, sequelae of Parkinson's disease,etc.

It has been reported that Alzheimer's disease is one of chronicpsychiatric disorders including disorders of higher cerebral functionssuch as loss of memory, impaired consciousness, spatiotemporal chaos,thinking ability, arithmetic ability, judgment, common sense, etc.Moreover, it has been reported that Alzheimer's disease also occurs inrelatively young people and most frequently occurs in elderly people asthe incidence is increased two times for each increase of 5 years in therange of 65 to 85 years old. While the pathogenesis of Alzheimer'sdisease is not clearly known, a decrease in acetylcholine function inthe central nervous system most commonly occurs, and thus therapeuticmethods of administering acetylcholine precursors or drugs that inhibitthe degradation of acetylcholine to increase the concentration ofacetylcholine in the brain have been used. Therefore,acetylcholinesterase (hereinafter, ‘AChE’) inhibitors have been usedalone or in combination with existing cholinesterase inhibitors, andexamples of such drugs include tacrine, donepezil, rivastigmine,galantamine, etc. While these drugs are acetylcholinesterase inhibitors,they only slow the progression of diseases, but have little effect onthe treatment and have limited therapeutic potential at the beginning ofthe disease, and thus made efforts have been made to develop drugs thattreat the underlying cause of Alzheimer's disease (Terry and Buccafusco,2003; Kar et al., 2004; Akhondzadeh et al., 2008; Cummings et al., 2008;Voss et al., 2008).

Vascular dementia is mostly caused by damage to the brain cells due tolack of blood supply to certain parts of the brain caused by cerebralarteriosclerosis. The causes of the vascular dementia and Alzheimer'sdisease are different, but they are the same in that they cause damageto memory and learning ability.

Accordingly, the present inventors have studied to develop an effectivemedicine for prevention or treatment of cognitive impairments such asdementia and found herbal extracts and their active ingredients whichcan effectively improve memory and learning ability, thus completing thepresent invention.

PRIOR ART LITERATURES Patent Literatures

-   KR 10-2010-0034779;-   KR 10-2004-0083414;-   KR 10-2003-0062963.

Non-Patent Literatures

-   2009 Alzheimer's disease facts and figures, Alzheimer's    Dement (2009) 5(3): p. 234-70;-   Whitehouse, P. J., Price, D. L., Struble, R. G., Clark, A. W.,    Coyle, J. T., and Delong, M. R. (1982) Alzheimer's disease and    senile dementia: loss of neurons in the basal forebrain. Science    215, 1237-12392;-   Hyman, B. T., Van Hoesen, G. W., Damasio, A. R., and    Barnes, C. L. (1984) Alzheimer's disease: cell-specific pathology    isolates the hippocampal formation. Science 225, 1168-11703.

DISCLOSURE Technical Problem

An object of the present invention is to provide a pharmaceuticalcomposition comprising spinosin for prevention or treatment of cognitiveimpairments.

Another object of the present invention is to provide a pharmaceuticalcomposition comprising a spinosin-containing herbal extract forprevention or treatment of cognitive impairments.

Still another object of the present invention is to provide a foodcomposition comprising spinosin for prevention or improvement ofcognitive impairments.

Yet another object of the present invention is to provide a foodcomposition comprising a spinosin-containing herbal extract forprevention or improvement of cognitive impairments.

Still yet object of the present invention is to provide a pharmaceuticalcomposition comprising spinosin for prevention or treatment ofdegenerative brain diseases.

A further object of the present invention is to provide a pharmaceuticalcomposition comprising a spinosin-containing herbal extract forprevention or treatment of degenerative brain diseases.

Another further object of the present invention is to provide a methodfor preventing or treatment of cognitive impairments or degenerativebrain diseases, the method comprising administering to a subject in needthereof a composition comprising spinosin.

Technical Solution

The present invention relates to a pharmaceutical composition comprisingspinosin for prevention or treatment of cognitive impairments.

Spinosin(6-(2-O-beta-D-Glocopyranosyl-beta-D-glucopyranosyl)-5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4H-1-benzopyran-4-one)is a compound represented by the following formula 1:

Spinosin represented by the above formula 1 can prevent or treatcognitive impairments. More particularly, the spinosin represented bythe above formula 1 can improve memory and learning ability, thuseffectively preventing and treating cognitive impairments.

The pharmaceutical composition comprising spinosin of the presentinvention can significantly improve learning ability, space perceptionability, and memory in animal models with memory impairment induced byscopolamine, and thus exhibits excellent activity in the prevention ortreatment of cognitive impairments and is particularly useful for theimprovement of dementia and amnesia.

Spinosin represented by the above formula 1 may be obtained from varioustypes of herbal medicines. For example, the spinosin represented by theabove formula 1 may be obtained from Zizyphus jujuba Mill var. inermis,Zizyphus jujuba Mill var. hoonensis, Zizyphus jujuba Mill var. spinosa,Passiflora edulis flavicarpa, Cayaponia tayuya, Desmodium tortuosum,Wilbrandia ebracteata, Strophioblachia fimbricalyx, Clutia abyssinica,Saccharopolyspora spinosa, or mixtures thereof, and may preferably beobtained from zizyphi spinosi semen, the seeds of Zizyphus jujuba Millvar. spinosa.

Spinosin represented by the above formula 1 may be obtained from anextract of the above-described herbal medicines or mixtures thereof, andthe spinosin represented by the above formula 1 may preferably beobtained from a water, methanol, ethanol, butanol, or hexane extract ofthe above-described herbal medicines or mixtures thereof. For example,the spinosin represented by the above formula 1 may be obtained from anextract of zizyphi spinosi semen, the seeds of Ziziphus jujuba Mill var.spinosa.

Preferably, the spinosin represented by the above formula 1 may beobtained from an extract of an ethanol extract of zizyphi spinosi semen,the seeds of Zizyphus jujuba Mill var. spinosa.

Otherwise, Spinosin represented by the above formula 1 may becommercially available.

According to the present invention, the cognitive impairments refer todiseases caused by impaired functions such as memory, space perceptionability, judgment, executive function, linguistic ability, etc. and mayinclude, for example, Alzheimer's disease, vascular dementia, otherdementias caused by various factors alcoholism, trauma, sequelae ofParkinson's disease, etc., or amnesia. Preferably, the cognitiveimpairment may be Alzheimer's disease.

The pharmaceutical composition comprising spinosin of the presentinvention can effectively inhibit scopolamine-induced memory impairmentsin mice even with a small amount.

The spinosin is an ingredient contained in the above-described herbalmedicines and can effectively prevent or treat cognitive impairmentswithout side effects.

In the pharmaceutical composition comprising spinosin for prevention ortreatment of cognitive impairments according to the present invention,the spinosin may be administered once or several times at a daily doseof about 1 mg to 120 mg for an adult, preferably at a daily dose of 30mg to 120 mg. However, the dose of the spinosin can be appropriatelyadjusted depending on the condition of a patient, such as the patient'sseverity, age, sex, weight, etc., and the formulation, administrationroute, and administration period.

The present invention relates to a pharmaceutical composition forprevention and treatment of cognitive impairments, comprising spinosinrepresented by the above formula 1 and an extract of Zizyphus jujubaMill var. inermis, Zizyphus jujuba Mill var. hoonensis, Zizyphus jujubaMill var. spinosa, Passiflora edulis flavicarpa, Cayaponia tayuya,Desmodium tortuosum, Wilbrandia ebracteata, Strophioblachia fimbricalyx,Clutia abyssinica, Saccharopolyspora spinosa, or mixtures thereof.

The above-mentioned Zizyphus jujuba Mill var. inermis, Zizyphus jujubaMill var. hoonensis, Zizyphus jujuba Mill var. spinosa, Passifloraedulis flavicarpa, Cayaponia tayuya, Desmodium tortuosum, Wilbrandiaebracteata, Strophioblachia fimbricalyx, Clutia abyssinica, andSaccharopolyspora spinosa comprise spinosin represented by the aboveformula 1. Therefore, the composition comprising an extract of theabove-described herbal medicines can prevent or treat cognitiveimpairments, like the spinosin, and has no side effects because it is aherbal extract.

The pharmaceutical composition of the preset invention may comprise anextract of Zizyphus jujuba Mill var. spinosa, preferably a water,hexane, ethanol, methanol, butanol, or the mixture thereof extract ofthe seeds of Ziziphus jujuba Mill var. spinosa, more preferably anethanol extract.

The spinosin-containing herbal extract can effectively prevent or treatcognitive impairments without side effects.

The herbal extract of the present invention comprises spinosin and thuscan significantly improve learning ability, space perception ability,and memory in animal models with memory impairment induced byscopolamine, and thus exhibits excellent activity in prevention ortreatment of cognitive impairments and is particularly useful for theimprovement of dementia and amnesia.

The pharmaceutical composition of the present invention may comprise theherbal extract in an amount of 0.1 to 50 wt % with respect to the totalweight of the composition. However, the content is not necessarilylimited thereto, but may vary depending on the patient's condition andthe type and progression of the disease.

In the pharmaceutical composition comprising a herbal extract forprevention or treatment of cognitive impairments according to thepresent invention, the herbal extract may be administered once orseveral times at a daily dose of about 10 mg to 1200 mg for an adult,preferably at a daily dose of 300 mg to 1200 mg. However, the dose ofthe herbal extract may be appropriately adjusted depending on thecondition of a patient, such as the patient's severity, age, sex,weight, etc., and the formulation, administration route, andadministration period.

The pharmaceutical composition comprising the spinosin represented bythe above formula 1 or the spinosin-containing herbal extract has notoxicity and side effects and thus can be used with confidence even whentaken for a long time for the purpose of prevention or treatment.

The present invention relates to a pharmaceutical composition comprisinga pharmaceutically acceptable salt of spinosin for prevention ortreatment of cognitive impairments or degenerative brain diseases. Theterm “pharmaceutically acceptable salt” refers to salts commonly used inthe pharmaceutical industry, such as inorganic ion salts formed withpotassium, sodium, magnesium, etc., inorganic acid salts formed withhydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodicacid, perchloric acid, tartaric acid, sulfuric acid, etc., organic acidsalts formed with acetic acid, trifluoroacetic acid, citric acid, maleicacid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaricacid, mandelic acid, propionic acid, lactic acid, glycolic acid,gluconic acid, galacturonic acid, glutamic acid, glutaric acid,glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillicacid, hydroiodic acid, etc., sulfonic acid salts formed withmethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, naphthalenesulfonic acid, etc., amino acid saltsformed with glycine, arginine, lysine, etc., and amine salts formed withtrimethylamine, triethylamine, ammonia, pyridine, picoline, etc., butthe types of salts used in the present invention are not limited to theabove-mentioned salts.

The cognitive impairments may be dementia or amnesia, and thedegenerative brain diseases may be Parkinson's disease, stroke, stroke,or Huntington's disease.

The pharmaceutical composition of the present invention may furthercomprise pharmaceutically acceptable additives such as diluents,binders, disintegrants, lubricants, pH-adjusting agents, antioxidants,and solubilizers within the range where effects of the present inventionare not impaired.

The diluents may include sugar, starch, microcrystalline cellulose,lactose (lactose hydrate), glucose, D-mannitol, alginate, alkaline earthmetal salt, clay, polyethylene glycol, calcium anhydrous hydrogenphosphate, and mixtures thereof; and the binders may include starch,microcrystalline cellulose, highly dispersive silica, mannitol,D-mannitol, sucrose, lactose hydrate, polyethylene glycol,polyvinylpyrrolidone (povidone), polyvinylpyrrolidone copolymer(copovidone), hypromellose, hydroxypropylcellulose, natural gum,synthetic gum, copovidone, gelatin, and mixtures thereof.

The disintegrants may include starches or modified starches such assodium starch glycolate, corn starch, potato starch, pregelatinizedstarch, etc.; clays such as bentonite, montmorillonite, veegum, etc.;celluloses such as microcrystalline cellulose, hydroxypropylcellulose,carboxymethylcellulose, etc.; algins such as sodium alginate, alginicacid, etc.; crosslinked celluloses such as croscarmellose sodium, etc.;gums such as guar gum, xanthan gum, etc.; crosslinked polymers such ascrosslinked polyvinylpyrrolidone (crospovidone), etc.; effervescentagents such as sodium bicarbonate, citric acid, etc.; and mixturesthereof.

The lubricants may include talc, stearic acid, magnesium stearate,calcium stearate, sodium lauryl sulfate, hydrogenated vegetable oil,sodium benzoate, sodium stearyl fumarate, glyceryl behenate, glycerylmonorate, glyceryl monostearate, glyceryl palmitostearate, colloidalsilicon dioxide, and mixtures thereof.

The pH-adjusting agents may include acidifying agents such as aceticacid, adipic acid, ascorbic acid, sodium ascorbate, sodium etherate,malic acid, succinic acid, tartaric acid, fumaric acid, and citric acid,and basifying agents such as precipitated calcium carbonate, aqueousammonia, meglumine, sodium carbonate, magnesium oxide, magnesiumcarbonate, sodium citrate, and tribasic calcium phosphate.

The antioxidants may include dibutyl hydroxy toluene, butylatedhydroxyanisole, tocopherol acetate, tocopherol, propyl gallate, sodiumhydrogen sulfite, and sodium pyrosulfite. The solubilizers that may beused in a prior-release compartment of the present invention may includesodium lauryl sulfate, polyoxyethylene sorbitan fatty acid esters suchas polysorbate, docusate sodium, poloxamer, etc.

For oral administration, the pharmaceutical composition comprisingspinosin or the spinosin-containing herbal extract according to thepresent invention may be formulated into solid dosage forms such astablets, pills, powders, granules, capsules, etc., and these soliddosage forms may be prepared by mixing spinosin or thespinosin-containing herbal extract with one or more excipients such asstarch, calcium carbonate, sucrose or lactose, gelatin, etc. Moreover,lubricants such as magnesium stearate, talc, etc. may be used inaddition to simple excipients. Furthermore, the pharmaceuticalcomposition may be formulated into liquid dosage forms such assuspensions, liquid for internal use, emulsions, syrups, etc., andvarious excipients such as humectants, sweeteners, aromatics,preservatives, etc. in addition to water and liquid paraffin may be usedfor the formulation of the liquid dosage forms.

For parenteral administration, the pharmaceutical composition comprisingspinosin or the spinosin-containing herbal extract according to thepresent invention may include sterile aqueous solutions, non-aqueoussolvents, suspensions, emulsions, lyophilized preparations, andsuppositories. Suitable non-aqueous solutions and suspensions mayinclude propylene glycol, polyethylene glycol, vegetable oils such asolive oil, and injectable esters such as ethyl oleate. Bases for thesuppositories may include Witepsol, macrogol, Tween 61, cacao butter,laurin butter, glycerogelatine, etc.

As used herein the term “administration” refers to the introduction ofthe composition for prevention and treatment of cognitive impairmentsaccording to the present invention to a patient in any appropriate way,and the composition for prevention and treatment of cognitiveimpairments according to the present invention may be administered viaany conventional administration route as long as the composition canreach a target tissue. For example, the composition may be administeredorally, intraperitoneally, intravenously, intramuscularly,subcutaneously, intradermally, intranasally, intrapulmonary, rectally,intracavitary, intraperitoneally, or intradurally, but not limitedthereto. For example, the composition may be administered by oral,rectal, or intravenous, intramuscular, subcutaneous, endometrial, orintracerebroventricular injection.

The pharmaceutical composition according to the present invention may beadministered once or several times a day at regular intervals.

The pharmaceutical composition according to the present invention mayfurther comprise other active ingredients effective for the treatment ofthe cognitive impairments.

The pharmaceutical composition according to the present invention may beused alone or in combination with various methods such as hormonetherapy, drug therapy, etc., for the prevention or treatment of thecognitive impairments.

The present invention relates to a food composition comprising spinosinrepresented by the above formula 1, a pharmaceutically acceptable saltthereof, or a spinosin-containing herbal extract for prevention orimprovement of the cognitive impairments.

The food composition according to the present invention may furthercomprise additives commonly used in food compositions, health functionalfoods, or beverages.

For example, the food composition of the present invention may comprisesweeteners such as white sugar, crystalline fructose, glucose,D-sorbitol, mannitol, isomaltooligosaccharide, stevioside, aspartame,acesulfame potassium, sucralose, etc., acidifiers such as anhydrouscitric acid, DL-malic acid, succinic acid, and salts thereof,preservatives such as benzoic acid and derivatives thereof, variousnutrients, vitamins, minerals (electrolyte), flavoring agents such assynthetic and natural flavoring agents, coloring agents and fillers(cheese, chocolate, etc.), pectic acid and salts thereof, alginic acidand salts thereof, organic acids, protective colloidal thickeners,pH-adjusting agents, stabilizers, preservatives, glycerin, alcohol,carbonators used in carbonated drinks, etc. Moreover, the foodcomposition of the present invention may comprise fruit pulp forpreparation of natural fruit juices and vegetable juices. This additivemay be used in an amount of less than about 20 parts by weight withrespect to 100 parts by weight of the food composition.

When the food composition of the present invention is a beverage, it mayfurther comprise flavoring agents or natural carbohydrates. Suitablenatural carbohydrates may include monosaccharides such as glucose andfructose, disaccharides such as maltose and sucrose, polysaccharidessuch as dextrin and cyclodextrin, and sugar alcohols such as xylitol,sorbitol, and erythritol. Moreover, suitable flavoring agents mayinclude natural flavoring agents such as thaumatin and stevia extracts(rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents suchas saccharin and aspartame. When the food composition is a beverage, thenatural carbohydrate may be contained in an amount of about 1 to 20 g,preferably about 5 to 12 g with respect to 100 mL of the composition.

The food composition comprising spinosin or the spinosin-containingherbal extract according to the present invention may be prepared in theform of powders, granules, tablets, capsules, or beverages and used asfoods, beverages, gums, teas, vitamin complexes, health supplementfoods, etc.

The composition comprising spinosin or the spinosin-containing herbalextract according to the present invention may be added to drugs, foods,beverages, etc. for prevention and treatment of the cognitiveimpairments. For example, the composition comprising spinosin or thespinosin-containing herbal extract according to the present inventionmay be added to food, beverages, gums, teas, vitamin complexes, healthsupplement foods, etc.

The food composition comprising spinosin or the spinosin-containingherbal extract according to the present invention may be added to foodsor beverages for prevention or improvement of the cognitive impairments.The composition of the present invention may be added in an amount of 1to 5 wt % with respect to the total weight of the food and may be addedin an amount of 0.02 to 10 g, preferably 0.3 g to 1 g, with respect to100 mL of the beverage.

The present invention provides a pharmaceutical composition comprisingspinosin for prevention or treatment of a degenerative brain diseases.The spinosin represented by the above formula 1 can effectively preventor treat cerebral infarction, stroke, Parkinson's disease, orHuntington's disease.

The present invention provides a pharmaceutical composition comprising aspinosin-containing herbal extract for prevention or treatment ofdegenerative brain diseases.

The spinosin-containing herbal extract can effectively prevent or treatcerebral infarction, stroke, Parkinson's disease, or Huntington'sdisease. The herbal extract may be obtained from Zizyphus jujuba Millvar. inermis, Zizyphus jujuba Mill var. hoonensis, Zizyphus jujuba Millvar. spinosa, Passiflora edulis flavicarpa, Cayaponia tayuya, Desmodiumtortuosum, Wilbrandia ebracteata, Strophioblachia fimbricalyx, Clutiaabyssinica, Saccharopolyspora spinosa, or mixtures thereof, and maypreferably be obtained from the seeds of Zizyphus jujuba Mill var.spinosa.

The present invention provides a method for preventing or treatment ofcognitive impairments or degenerative brain diseases, the methodcomprising administering to a subject in need thereof a compositioncomprising spinosin or a pharmaceutically acceptable salt thereof. Thecognitive impairments may be dementia or amnesia, and the degenerativebrain diseases may be cerebral infarction, stroke, Parkinson's disease,or Huntington's disease.

The present invention provides a method for preventing or treatment ofcognitive impairments or degenerative brain diseases, the methodcomprising administering to a subject in need thereof a compositioncomprising a spinosin-containing herbal extract. The herbal extract maybe obtained from Zizyphus jujuba Mill var. inermis, Zizyphus jujuba Millvar. hoonensis, Zizyphus jujuba Mill var. spinosa, Passiflora edulisflavicarpa, Cayaponia tayuya, Desmodium tortuosum, Wilbrandiaebracteata, Strophioblachia fimbricalyx, Clutia abyssinica,Saccharopolyspora spinosa, or mixtures thereof, and may preferably beobtained from the seeds of Zizyphus jujuba Mill var. spinosa.

Advantageous Effects

The pharmaceutical composition comprising spinosin or aspinosin-containing herbal extract according to the present inventioncan effectively prevent or treat cognitive impairments. Specifically,the pharmaceutical composition comprising spinosin or thespinosin-containing herbal extract according to the present inventioncan significantly improve memory and learning ability, thus effectivelyprevent or treat dementia.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the effect of a pharmaceutical composition comprisingspinosin according to the present invention on the improvement of memoryand learning ability.

FIGS. 2 to 5 are shows the effect of a pharmaceutical compositioncomprising a spinosin-containing zizyphi spinosi semen extract accordingto the present invention on the improvement of memory and learningability.

MODE FOR INVENTION

Hereinafter, the present invention will be described in detail withreference to the following Examples and Experimental Examples. However,the following Examples and Experimental Examples are merely illustrativeof the present invention and the present invention is not limited by thefollowing Examples and Experimental Examples.

Moreover, all reagents and solvents mentioned below were purchased fromSigma unless otherwise stated, and optical rotations were measured usinga JASCO P-1020 polarimeter. UV spectra were measured using a HitachiJP/U3010, IR spectra were measured using a JASCO FT/IR-5300, NMR spectrawas measured using a Bruker Avance 400 (400 MHz), and FAB Mass spectrawere measured using a JEOL JMS-700 mass spectrometer.

Example 1 Preparation of Spinosin-Containing Zizyphi Spinosi SemenExtract

12 kg of zizyphi spinosi semen (the seeds of Ziziphus jujuba Mill var.spinosa Hu ex H. F. Chou) was crushed by a crusher and distributed intoextraction bottles, n-hexane was added to the crushed zizyphi spinosisemen until it covered the surface of the crushed zizyphi spinosi semen,and the mixture was left to stand at room temperature for 3 days andthen filtered. The hexane extracts were combined and concentrated whenthe hexane solutions were transparent after the same method was repeatedfour times.

After the removal of the hexane extracts, 70% ethanol was added to theremaining zizyphi spinosi semen until it covered the surface of zizyphispinosi semen, and the mixture was left to stand at room temperature for3 days and then filtered and concentrated under reduced pressure. 70%(v/v) ethanol was added to the residue and then extracted in water bath,followed by filtration and concentration. The extraction was performed 6times by the method of adding 70% (V/v) ethanol and then extraction inwater bath and filtration, and all the extracts were combined andconcentrated under reduced pressure, yielding 70% (v/v) ethanol softext. of zizyphi spinosi semen.

Example 2 Isolation and Purification of Spinosin

n-hexane, methanol, and water were added at a volume ratio of 10:9:1 tothe ethanol soft ext. of zizyphi spinosi semen prepared in the aboveExample 1, the mixture was shaken and left to stand overnight to removethe hexane soluble fraction, and then the 90% methanol fraction wasconcentrated under reduced pressure. Distilled water was added to theconcentrated 90% (v/v) methanol fraction, ethyl acetate in an amountequivalent to the distilled water was added, and then the ethyl acetatefraction was removed. n-butanol of an equivalent amount was added to thewater layer, and then the n-butanol fraction was concentrated underreduced pressure.

The n-butanol fraction obtained by concentration under reduced pressurewas loaded onto silica gel column and subjected to chromatography(7:1:0.5→7:1.5:0.5→7:2:0.5→MeOH) with solutions of methylene chloride,methanol, and water at volume ratios of 7:1:0.5, 7:1.5:0.5, and 7:2:0.5and methanol as an eluent to obtain thirty small fractions, and the25^(th) small fraction containing spinosin was obtained from the thirtysmall fractions by performing TLC.

The 25^(th) small fraction was loaded onto silica gel column andsubjected to chromatography (100:8:6→100:10:7.5→100:12:9) with solutionsof ethyl acetate, methanol, and water at volume ratios of 100:8:6,100:10:7.5, 100:12:9 as an eluent to obtain small fractions, and the3^(rd) small fraction containing spinosin was obtained from the smallfractions by performing TLC.

The 3^(rd) small fraction was recrystallized with methanol to yieldspinosin (3.2 g) as a pale yellow amorphous powder (yield: 0.027%).

The physicochemical properties of the spinosin were as follows:

TLC Rf: 0.27 (absorbent: silica gel GF₂₅₄, developing solvent:chloroform/methanol/water (520:280:80), color reagent: 20% sulfuric acidsolution)

Melting point: 237-240° C.

[α]_(D) ²⁴=−47.5° (c=0.01, MeOH)

UV, λ_(max) (log ε) (MeOH) 216 (sh, 4.74), 272 (4.46), 334 (4.52) nm

IR, u_(max) 3141 (OH), 1649, 1603, 1484, 1453, 1347, 1196, 1054, 1020,841 cm⁻¹

¹H-NMR (400 MHz, DMSO-d₆+D₂O) δ: 2.56 (1H, dt, J=9.0 Hz, H-5′″), 2.74(1H, dt, J=9.4 Hz, H-5′″), 2.83 (2H, t, J=8.3, H-2′″), 2.93 (2H, br d,9.4 Hz, H-6′″), 2.94, 2.99 (1H each, t, J=8.5 Hz, H-4′″), 3.39 (2H, brd, J=12.1 Hz, H-6″), 3.70 (2H, t, J=9.0 Hz, H-3′″), 3.88 (6H, s, OCH₃),4.15 (2H, d, J=7.8, H-1′″), 4.28, 4.45 (1H each, t, J=9.3 Hz, H-2″),4.68 (2H, d, J=9.8 Hz, H-1″), 6.77, 6.79 (1H each, s, H-8), 6.80 (2H, s,H-3), 6.94 (4H, d, J=8.6 Hz, H-3′,5′), 7.95 (4H, d, J=8.6 Hz, H-2′,6′),13.5, 13.6 (1H each, s, 5-OH)

¹³C-NMR (100 MHz, DMSO-d₆+D₂O) δ: 56.5, 56.9 (OCH₃), 60.2, 60.8 (C-6′″),61.6 (C-6″), 69.3, 69.6 (C-4′″), 70.6 (C-4″), 71.1, 71.4 (C-1″), 74.7,74.8 (0-2′″), 76.4 (C-3′″), 76.6, 76.8 (C-5′″), 78.4, 78.7 (C-3″), 80.9,81.3 (C-2″), 81.7, 82.0 (C-5″), 90.8, 91.3 (C-8), 103.4, 103.5 (C-3),104.5, 104.7 (C-10), 105.4, 105.6 (C-1′″), 108.8 (C-6), 116.4 (C-3′,5′),121.4, 121.5 (C-1′), 128.9 (C-2′,6′), 157.4, 157.5 (C-9), 159.6, 160.5(C-5), 161.4 (0-4′), 164.2 (C-2), 164.3, 165.4 (C-7), 182.3, 182.6 (C-4)

Positive FAB-MS m/z 609 [M+H]⁺

Experimental Example

Determination of Effect on Dementia Treatment

Experiments were performed using models with memory impairment inducedby scopolamine to determine the effect of the spinosin and the zizyphispinosi semen extract containing spinosin as an active ingredientprepared in Examples 1 and 2, and the detailed experimental methods areas follows:

1) Preparation of Experimental Animals

6-week-old ICR mice (Orient Bio Inc., Korea), weighing about 26 to 28 g,were fed with water and food freely available for 5 days in anenvironment, where the temperature was about 23±2° C., the humidity wasabout 55±10%, and the light-dark cycle was 12 hours (Animal Laboratoryat the College of Pharmacy in Kyung Hee university), and used in theexperiments.

2) Statistical Analysis

Statistical analysis of all experimental data was performed using aone-way analysis of variance (ANOVA), and when the statisticalsignificance was recognized, the significance was assessed at P<0.05using Student-Newman-Keuls Test.

3) Experimental Example 1 Passive Avoidance Test 1

A passive avoidance test apparatus was prepared for the experiment. Thepassive avoidance test apparatus was divided into a first space and asecond space, a guillotine door was placed between the two spaces, andthe first and second spaces are connected by the door. The first spacewas kept light using illumination, and the second space was kept dark.The floor of the second space kept dark was provided with a grid, and anelectric shock of 0.5 mA was applied for 3 seconds through the grid onthe floor when the experimental animal moved to the dark space.

The mice prepared in the above section 1) were divided into 7 groupsincluding drug administration groups 1 to 4, to which spinosin was to beadministered, and control groups 1 to 3 (10 mice per group). Thespinosin of Example 2 was dissolved in 10% Tween 80 (Polyoxyethylenesorbitan monooleate: Sigma, U.S.A.) and administered to drugadministration groups 1 to 4 at doses of 2.5 mg/Kg, 5 mg/Kg, 10 mg/Kg,and 20 mg/Kg. Meanwhile, donepezil (Sigma-Aldrich Chemistry Co.) wasadministered to control group 1 at a dose of 5 mg/Kg, and 10% Tween 80was administered to control groups 2 and 3 at a dose of 0.15 mL.

After about 30 minutes, scopolamine (Sigma-Aldrich Chemistry Co.)dissolved in distilled water was intraperitoneally administered to drugadministration groups 1 to 4 and control groups 1 and 2 at a dose of 1mg/Kg (Ebert, U. et al., Eur. J. Clin. Invest., 28, pp 944-949, 1998),and 0.9% saline solution was intraperitoneally administered to controlgroup 3. After 30 minutes, learning about the passive avoidance testapparatus was performed on the mice in drug administration groups 1 to 4and control groups 1 to 3, respectively. Specifically, the mice wereplaced in the first space kept light, and after a seek time of about 20seconds, the guillotine door was opened, and the latency time taken forthe mice to move to the second space kept dark was measured.

The mice that did not move to the second space kept dark until 60seconds elapsed after the guillotine door was opened were excluded fromthe experiment.

After 24 hours since the learning was completed, the present experimentwas performed on drug administration groups 1 to 4 and control groups 1to 3, respectively. The latency time taken for all four feet of themouse to enter the dark space after the guillotine door was opened fromthe seek time of 10 seconds was measured up to 300 seconds. A longerlatency time represents that the learning and memory of the passiveavoidance is better.

In learning and in the present experiment, the average latency times ofthe mice in each group are shown in the following Table 1 and FIG. 1:

TABLE 1 Present Experiment Experimental Group Learning (sec) (sec) Drugadministration 25.88 ± 13.72 59.13 ± 46.88 group 1 (2.5 mg/Kg) Drugadministration 29.38 ± 18.17 107.1 ± 91.70 group 2 (5 mg/Kg) Drugadministration 20.50 ± 9.196 131.3 ± 69.94 group 3 (10 mg/Kg) Drugadministration 36.00 ± 16.45 146.0 ± 83.53 group 4 (20 mg/Kg) Controlgroup 1 25.13 ± 13.68 151.9 ± 37.24 (donepezil) Control group 2 26.50 ±18.16 41.33 ± 18.92 Control group 3 29.17 ± 18.15 300

As can be seen from Table 1 and FIG. 1, the latency time taken for themice in drug administration groups 1 to 4, to which the spinosinprepared in Example 2 was administered, to move the second space wassignificantly increased compared to that in control group 2 to whichonly scopolamine was administered. Specifically, the latency time indrug administration group 1, to which the spinosin was administered at adose of 2.5 mg/Kg, was increased about 20 seconds compared to that incontrol group 2, and the latency time in drug administration group 2, towhich the spinosin was administered at a dose of 5 mg/Kg, was increasedabout 2.5 times compared to that in control group 2. Moreover, thelatency time in drug administration group 3, to which the spinosin wasadministered at a dose of 10 mg/Kg, was increased about 3 times,compared to that in control group 2, and the latency time in drugadministration group 4, to which the spinosin was administered at a doseof 20 mg/Kg, was increased about 3.5 times compared to that in controlgroup 2. Particularly, it could be seen that the memory and learningability of the mice with memory impairment induced by scopolamine indrug administration groups 3 and 4 was improved to an extent similar tothat in control group 1 to which donepezil was administered.

It can be seen from these results that the spinosin prepared in Example2 can effectively prevent or treat cognitive impairments such asdementia.

4) Experimental Example 2 Passive Avoidance Teat 2

The passive avoidance test was performed on drug administration groups 1to 4 and control groups 1 to 3 by the same method as ExperimentalExample 1, except that the zizyphi spinosi semen extract extracted with70% ethanol in Example 1 was dissolved in 10% Tween 80 and administeredto drug administration groups 1 to 4 at doses of 25 mg/kg, 50 mg/kg, 100mg/kg, and 200 mg/kg.

The results of the experiment are shown in the following Table 2 andFIG. 2:

TABLE 2 Present Experiment Experimental Group Learning (sec) (sec) Drugadministration 25.75 ± 15.92 49.50 ± 46.26 group 1 (25 mg/Kg) Drugadministration 23.25 ± 18.17 40.00 ± 17.51 group 2 (50 mg/Kg) Drugadministration 32.13 ± 18.92 173.9 ± 77.84 group 3 (100 mg/Kg) Drugadministration 27.75 ± 19.00 112.8 ± 89.67 group 4 (200 mg/Kg) Controlgroup 1 17.25 ± 5.365 140.5 ± 25.47 (donepezil) Control group 2 32.80 ±21.26 26.10 ± 25.27 Control group 3 21.00 ± 8.380 228.7.00 ± 50.65  

As can be seen from Table 2 and FIG. 2, the latency time taken for themice in drug administration groups 1 to 4, to which thespinosin-containing zizyphi spinosi semen extract prepared in Example 1was administered, to move the second space was significantly increasedcompared to that in control group 2 to which only scopolamine wasadministered. Specifically, the latency time in drug administrationgroups 1 and 2, to which the spinosin was administered at doses of 25mg/Kg and 50 mg/Kg, respectively, was increased about 2 times comparedto that in control group 2. Moreover, the latency time in drugadministration group 3, to which the spinosin was administered at a doseof 100 mg/Kg, was increased about 6 times compared to that in controlgroup 2, and the latency time in drug administration group 4, to whichthe spinosin was administered at a dose of 200 mg/Kg, was increasedabout 4.3 times compared to that in control group 2. Particularly, itcould be seen that the memory and learning ability of the mice withmemory impairment induced by scopolamine in drug administration groups 3and 4 was improved to an extent similar to that in control group 1 towhich donepezil was administered.

It can be seen from these results that the spinosin-containing zizyphispinosi semen extract prepared in Example 1 can effectively prevent ortreat cognitive impairments such as dementia.

5) Experimental Example 3 Y-Maze Test

A Y-maze was prepared for the experiment. The Y-maze had three arms, inwhich each arm was 42 cm in length, 3 cm in width, and 12 cm in height,the angle formed by three arms was 120 degrees, and the Y-maze was madeof black polyvinyl resin.

The mice prepared in the above section 1) were divided into 5 groupsincluding drug administration groups 1 and 2, to which the zizyphispinosi semen extract prepared in Example 1 was to be administered, andcontrol groups 1 to 3 (10 mice per group).

160 mg of the zizyphi spinosi semen extract of Example 1 was dissolvedin 4 mL of 10% Tween 80 (Polyoxyethylene sorbitan monooleate: Sigma,U.S.A.) and administered to drug administration groups 1 and 2 at dosesof 100 mg/Kg and 200 mg/Kg. Meanwhile, donepezil (Sigma-AldrichChemistry Co.) was administered to control group 1 at a dose of 5 mg/Kg,and 10% Tween 80 was administered to control groups 2 and 3 at a dose of0.15 mL.

After about 30 minutes, scopolamine (Sigma-Aldrich Chemistry Co.)dissolved in distilled water was intraperitoneally administered to drugadministration groups 1 and 2 and control groups 1 and 2 at a dose of 1mg/Kg (Ebert, U. et al., Eur. J. Clin. Invest., 28, pp 944-949, 1998),and 0.9% saline solution was intraperitoneally administered to controlgroup 3.

After 30 minutes, the mice in drug administration groups 1 and 2 andcontrol groups 1 to 3 were carefully placed on one of three arms of theY-maze, divided into A, B, and C, respectively, and allowed to freelymove, and then the arms where the mice entered were recorded. At thistime, only the case where the mouse' tail fully entered the arm wasrecorded as the arm where the experimental animal entered, and the casewhere the mouse entered the same arm was also recorded.

1 point was given to the case where the mouse sequentially entered threedifferent arms (actual alternation). Alternation behavior was defined asthe case where the mouse entered all three arms and was converted into apercentage (%) by the following Formula 1 (Sarter, M. et al.,Psychopharmacology., 94, pp 491-495, 1998).

$\begin{matrix}{{{Alternatoin}\mspace{14mu} {{behavior}(\%)}} = {\frac{{Actual}\mspace{14mu} {alternation}}{{Maximum}\mspace{14mu} {alternation}} \times 100}} & \left\lbrack {{Formula}\mspace{14mu} 1} \right\rbrack\end{matrix}$

(Maximum alternation: Total entry—2)

The alternation behaviors converted by the above Formula 1 are shown inthe following Table 3 and FIG. 3, and the total entries, eachrepresenting the total number of entries, are shown in the followingTable 4 and FIG. 4:

TABLE 3 Experimental Group Alternation behavior (%) Drug administrationgroup 1 63.32 ± 9.005 (100 mg/Kg) Drug administration group 2 60.91 ±6.369 (200 mg/Kg) Control group 1 (Donepezil) 65.71 ± 10.19 Controlgroup 2 50.92 ± 14.59 Control group 3 76.53 ± 8.379

TABLE 4 Experimental Group Total entry Drug administration group 1 37.00± 14.30 (100 mg/Kg) Drug administration group 2 32.30 ± 7.675 (200mg/Kg) Control group 1 (Donepezil) 34.75 ± 7.265 Control group 2 24.75 ±6.519 Control group 3 26.88 ± 13.78

As can be seen from Table 3 and FIG. 3, the alternation behaviors indrug administration groups 1 and 2, to which the spinosin-containingzizyphi spinosi semen extract prepared in Example 1 was administered,were increased compared to those in control group 2, to which onlyscopolamine was administered, and were similar to those in control group1 to which donepezil was administered. Moreover, as can be seen fromTable 4 and FIG. 4, the total entries in drug administration groups 1and 2 and control groups 1 to 3 were all similar to each other.Therefore, it could be seen that the increase in the alternationbehavior did not result from the change in the activity of the mice, andthus it could be seen that the memory and learning ability of the micein drug administration groups 1 and 2 was significantly improved by thespinosin-containing zizyphi spinosi semen extract prepared in Example 1.

It can be seen from these results that the spinosin-containing zizyphispinosi semen extract prepared in Example 1 can effectively prevent ortreat cognitive impairments such as dementia.

6) Experimental Example 4 Morris Water Maze Test

A Morris water maze test apparatus was prepared for the experiment. Fourlabels such as star, square, triangle, and circle were put at regularintervals on a circular pool of 90 cm in diameter and 45 cm in height,and a platform of 29 cm in height was positioned below the star. Waterwas filled up to 0.5 cm from the platform (water temperature: 21±1° C.)and became cloudy with pigment such that the platform was not visiblefrom the surface of the water.

The mice prepared in the above section 1) were divided into 5 groupsincluding drug administration groups 1 and 2, to which the zizyphispinosi semen extract prepared in Example 1 was to be administered, andcontrol groups 1 to 3 (10 mice per group).

160 mg of the zizyphi spinosi semen extract of Example 1 was dissolvedin 4 mL of 10% Tween 80 (Polyoxyethylene sorbitan monooleate: Sigma,U.S.A.) and administered to drug administration groups 1 and 2 at dosesof 100 mg/Kg and 200 mg/Kg. Meanwhile, donepezil (Sigma-AldrichChemistry Co.) was administered to control group 1 at a dose of 5 mg/Kg,and 10% Tween 80 was administered to control groups 2 and 3 at a dose of0.15 mL.

After about 30 minutes, scopolamine (Sigma-Aldrich Chemistry Co.)dissolved in distilled water was intraperitoneally administered to drugadministration groups 1 and 2 and control groups 1 and 2 at a dose of 1mg/Kg (Ebert, U. et al., Eur. J. Clin. Invest., 28, pp 944-949, 1998),and 0.9% saline solution was intraperitoneally administered to controlgroup 3.

After 30 minutes, the time taken for the mice, put down in one sectionof the pool, in drug administration groups 1 and 2 and control groups 1to 3 to reach the platform was measured for 60 seconds. After 30minutes, the time taken for the mice, repositioned to the originalposition, to reach the platform was measured until 60 seconds. Theabove-described process was repeated 4 times, and the average valueswere obtained. The experiment was performed in the same manner as on thefirst day while administering the drugs for 4 days, except that theposition where the mice were first put down was changed.

The results of the experiment are shown in the following Table 5 andFIG. 5:

TABLE 5 Experimental Time (sec) group 1^(st) day 2^(nd) day 3^(rd) day4^(th) day Drug 56.10 ± 9.088  59.85 ± 0.4743 49.85 ± 16.64 46.70 ±15.96 administration group 1 (100 mg/Kg) Drug 52.75 ± 12.17 46.20 ±12.52 43.45 ± 18.19 30.55 ± 20.45 administration group 2 (200 mg/Kg)Control group 1 59.34 ± 2.055 48.75 ± 14.98 42.80 ± 15.16 34.35 ± 13.80(Donepezil) Control group 2 57.75 ± 7.115 57.05 ± 6.693 53.95 ± 11.1357.30 ± 6.701 Control group 3 57.10 ± 6.736 40.35 ± 14.98 27.65 ± 16.2725.60 ± 13.70

As can be seen from Table 5 and FIG. 5, the time taken for the mice indrug administration groups 1 and 2, to which the spinosin-containingzizyphi spinosi semen extract prepared in Example 1 was administered, toreach the platform was reduced by more than 10 seconds and 25 seconds,respectively, on the 3^(rd) and 4^(th) day compared to that in controlgroup 2 to which only scopolamine was administered. Particularly, in thecase of drug administration group 2, the time taken for the mice toreach the platform was reduced compared to that in control group 1 towhich donepezil was administered, indicating that thespinosin-containing zizyphi spinosi semen extract prepared in Example 1had superior memory and learning ability to the donepezil.

Preparation of Formulations and Food Compositions

Preparation of Powders

Powders were prepared by mixing 20 mg of the spinosin-containing zizyphispinosi semen extract prepared in Example 1, 100 mg of lactose, and 10mg of talc and packing the mixture in airtight bags.

Preparation of Tablets

Tablets were prepared using the spinosin-containing zizyphi spinosisemen extract prepared in Example 1 and the ingredients shown in thefollowing Table 6:

TABLE 6 Ingredients Content (mg) Zizyphi spinosi semen extract 10(Example 1) Corn starch 100 Lactose 100 Magnesium stearate 2

Tablets were prepared by mixing the above ingredients and compressingthe mixture into tablets according to a conventional method forpreparing tablets.

Preparation of Capsules

TABLE 7 Ingredients Content (mg) Zizyphi spinosi semen extract 10(Example 1) Crystalline cellulose 100 Lactose 100 Magnesium stearate 0.2

Capsules were prepared by mixing the above ingredients and filling themixture in gelatin capsules according to a conventional method forpreparing capsules.

Preparation of Injections

TABLE 8 Ingredients Content (mg) Zizyphi spinosi semen extract 10(Example 1) Mannitol 180 Sterile distilled water for 2974 injectionNa₂HPO₄•12H₂O 26

Injections were prepared by mixing the above ingredients to prepare asolution and filling the solution in ampoules (2 mL), followed bysterilization, according to a conventional method for preparinginjections.

Preparation of Liquid Formulations

TABLE 9 Ingredients Content Zizyphi spinosi semen extract 20 mg(Example 1) Isomerized sugar 10 g Mannitol 5 g Purified water 85 ml

Liquid formulations were prepared by dissolving the above ingredients inpurified water, adding a suitable amount of lemon flavor, addingpurified water to the mixture to prepare a solution of 100 ml, andloading the solution into brown bottles, followed by sterilization,according to a conventional method for preparing liquid formulations.

Preparation of Health Foods

TABLE 10 Ingredients Content Zizyphi spinosi semen extract 1000 mg(Example 1) Nicotinic acid amide 1.7 mg Folic acid 50 μg Calciumpantothenate 0.5 mg Ferrous sulfate 1.75 mg Zinc oxide 0.82 mg Magnesiumcarbonate 25.3 mg Potassium Phosphate monobasic 15 mg calcium Phosphatedibasic 55 mg Potassium citrate 90 mg Calcium carbonate 100 mg Magnesiumchloride 24.8 mg Vitamin mixture Suitable amount Mineral mixtureSuitable amount

Health food compositions were prepared according to a conventionalmethod by mixing the above ingredients according to a conventionalmethod for preparing health foods and then preparing granules.

Preparation of Health Beverages

TABLE 11 Ingredients Content Zizyphi spinosi semen extract 1000 mg(Example 1) Citric acid 1000 mg Oligosaccharide 100 g Japanese apricotconcentrate 2 g Taurine 1 g Purified water Total 900 mL

Health beverages were prepared by dissolving the above ingredients inpurified water to prepare a solution of a 900 mL, stirring and heatingthe solution at 85° C. for about 1 hour, filtering the solution, fillingthe solution in sterilized 2 L bottles, sealing and sterilizing thebottles, and keeping the bottles under refrigeration according to aconventional method for preparing health beverages.

1. A pharmaceutical composition comprising spinosin for prevention ortreatment of cognitive impairments.
 2. The pharmaceutical composition ofclaim 1, wherein the spinosin is obtained from at least one extractselected from the group consisting of Zizyphus jujuba Mill var. inermis,Zizyphus jujuba Mill var. hoonensis, Zizyphus jujuba Mill var. spinosa,Passiflora edulis flavicarpa, Cayaponia tayuya, Desmodium tortuosum,Wilbrandia ebracteata, Strophioblachia fimbricalyx, Clutia abyssinica,and Saccharopolyspora spinosa.
 3. The pharmaceutical composition ofclaim 1, wherein the cognitive impairments comprise dementia andamnesia.
 4. A pharmaceutical composition comprising spinosin forprevention or treatment of cognitive impairments, wherein thepharmaceutical composition comprises at least one extract selected fromthe group consisting of Zizyphus jujuba Mill var. inermis, Zizyphusjujuba Mill var. hoonensis, Zizyphus jujuba Mill var. spinosa,Passiflora edulis flavicarpa, Cayaponia tayuya, Desmodium tortuosum,Wilbrandia ebracteata, Strophioblachia fimbricalyx, Clutia abyssinica,and Saccharopolyspora spinosa.
 5. The pharmaceutical composition ofclaim 4, wherein the extract is obtained from the seeds of Ziziphusjujuba Mill var. spinosa.
 6. A food composition comprising spinosin forprevention or treatment of cognitive impairments.
 7. A food compositioncomprising spinosin for prevention or improvement of cognitiveimpairments, wherein the food composition comprises at least one extractselected from the group consisting of Zizyphus jujuba var. inermis,Zizyphus jujuba var. hoonensis, Zizyphus jujuba var. spinosa, Passifloraedulis flavicarpa, Cayaponia tayuya, Desmodium tortuosum, Wilbrandiaebracteata, Strophioblachia fimbricalyx, Clutia abyssinica, andSaccharopolyspora spinosa.